Fusion proteins

Abstract

A single chain, polypeptide fusion protein, comprising: a non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent; a dynorphin Targeting Moiety that is capable of binding to a Binding Site on the nociceptive sensory afferent, which Binding Site is capable of undergoing endocytosis to be incorporated into an endosome within the nociceptive sensory afferent; a protease cleavage site at which site the fusion protein is cleavable by a protease, wherein the protease cleavage site is located between the non-cytotoxic protease or fragment thereof and the dynorphin Targeting Moiety; and a translocation domain that is capable of translocating the protease or protease fragment from within an endosome, across the endosomal membrane and into the cytosol of the nociceptive sensory afferent. Nucleic acid sequences encoding the polypeptide fusion proteins, methods of preparing same and uses thereof are also described.

What is claimed is: 
     
       1. A single chain, polypeptide fusion protein, comprising:
 a. a non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent; 
 b. a Targeting Moiety that is capable of binding to a Binding Site on the nociceptive sensory afferent, which Binding Site is capable of undergoing endocytosis to be incorporated into an endosome within the nociceptive sensory afferent; 
 c. a protease cleavage site at which site the fusion protein is cleavable by a protease, wherein the protease cleavage site is located between the non-cytotoxic protease or fragment thereof and the Targeting Moiety; 
 d. a translocation domain that is capable of translocating the protease or protease fragment from within an endosome, across the endosomal membrane and into the cytosol of the nociceptive sensory afferent; 
 wherein the Targeting Moiety is located between the protease cleavage site and the translocation domain, and wherein
 (i) the non-cytotoxic protease component forms a disulphide bond with the translocation component of the fusion protein, wherein the amino acid residue of the protease component that forms the disulphide bond is located within the last 20 C-terminal amino acid residues of the protease component, and the amino acid residue within the translocation component that forms the second part of the disulphide bond is located within the first 20 N-terminal amino acid residues of the translocation component; or 
 (ii) the non-cytotoxic protease component forms a disulphide bond with the Targeting Moiety component of the fusion protein, wherein the amino acid residue of the protease component that forms the disulphide bond is located within the last 20 C-terminal amino acid residues of the protease component, and the amino acid residue within the Targeting Moiety component that forms the second part of the disulphide bond is located within the last 20 C-terminal amion acid residues of the Targeting Moiety component. 
 
 
     
     
       2. The fusion protein according to  claim 1 , wherein the Targeting Moiety and the protease cleavage site are separated by at most 10 amino acid residues. 
     
     
       3. The fusion protein according to  claim 1 , wherein the Targeting Moiety and the protease cleavage site are separated by at most 5 amino acid residues. 
     
     
       4. The fusion protein according to  claim 1 , wherein the Targeting Moiety and the protease cleavage site are separated by zero amino acid residues. 
     
     
       5. The fusion protein according to  claim 1 , wherein the non-cytotoxic protease is a clostridial neurotoxin L-chain or an IgA protease. 
     
     
       6. The fusion protein according to  claim 1 , wherein the translocation domain is the H N  domain of a clostridial neurotoxin. 
     
     
       7. The fusion protein according to  claim 1 , wherein the Targeting Moiety comprises at most 50 amino acid residues. 
     
     
       8. The fusion protein according to  claim 1 , wherein the Targeting Moiety comprises at most 40 amino acid residues. 
     
     
       9. The fusion protein according to  claim 1 , wherein the Targeting Moiety comprises at most 30 amino acid residues. 
     
     
       10. The fusion protein according to  claim 1 , wherein the Targeting Moiety comprises at most 20 amino acid residues. 
     
     
       11. The fusion protein according to  claim 1 , wherein the Targeting Moiety:
 (i) is an opioid; 
 (ii) is an agonist of a receptor present on a nociceptive sensory afferent; and 
 (iii) binds to the ORL 1  receptor. 
 
     
     
       12. The fusion protein according to  claim 11 , wherein the Targeting Moiety is an agonist of a receptor present on a primary nociceptive sensory afferent. 
     
     
       13. The fusion protein according to  claim 11 , wherein the Targeting Moiety binds specifically to the ORL 1  receptor. 
     
     
       14. The fusion protein according to  claim 11 , wherein the Targeting Moiety is an agonist of the ORL 1  receptor. 
     
     
       15. The fusion protein according to  claim 1 , wherein the Targeting Moiety has at least 70% sequence identity to SEQ ID NO:38 or a fragment thereof, or wherein the Targeting Moiety has at least 80% sequence identity to SEQ ID NO:38 or a fragment thereof, or wherein the Targeting Moiety has at least 90% sequence identity to SEQ ID NO:38 or a fragment thereof, or wherein the Targeting Moiety has at least 95% sequence identity to SEQ ID NO:38 or a fragment thereof, or wherein the Targeting Moiety is SEQ ID NO: 38 or a fragment thereof, or wherein the Targeting Moiety is one of SEQ ID NOs:40, 42, 44, 46, 48 or 50, or wherein the Targeting Moiety is nociceptin. 
     
     
       16. The fusion protein according to  claim 1 , wherein the Targeting Moiety has at least 70% sequence identity to SEQ ID NO: 89 or a fragment thereof, or wherein the Targeting Moiety has at least 80% sequence identity to SEQ ID NO:89 or a fragment thereof, or wherein the Targeting Moiety has at least 90% sequence identity to SEQ ID NO:89 or a fragment thereof, or wherein the Targeting Moiety has at least 95% sequence identity to SEQ ID NO:89 or a fragment thereof, or wherein the Targeting Moiety is SEQ ID NO:89, or wherein the Targeting Moiety is dynorphin. 
     
     
       17. The fusion protein according to  claim 1 , wherein the Targeting Moiety is selected from the group consisting of nociceptin, β-endorphin, endomorphine-1, endomorphine-2, dynorphin, met-enkephalin, leu-enkephalin, galanin, and PAR-2 peptide. 
     
     
       18. The fusion protein according to  claim 1 , wherein the fusion protein comprises a purification tag. 
     
     
       19. The fusion protein according to  claim 1 , wherein the fusion protein comprises a purification tag which is present at the N-terminal and/or C-terminal end of the fusion protein. 
     
     
       20. The fusion protein according to  claim 1 , wherein the fusion protein comprises a purification tag and wherein the purification tag is joined to the fusion protein by a peptide spacer molecule. 
     
     
       21. The fusion protein according to  claim 1 , wherein the translocation domain is separated from the Targeting Moiety by a peptide spacer molecule. 
     
     
       22. A polypeptide fusion protein according to  claim 1 , comprising any one of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 52, 59, 61, 64, 67, 69, 71, 73, 76, 79, 82, 85, 88, 91, 92, 93, 94, 95 96 or 97. 
     
     
       23. A nucleic acid sequence encoding the polypeptide fusion protein according to  claim 1 . 
     
     
       24. A nucleic acid sequence according to  claim 23  wherein the nucleic acid sequence comprises any one of SEQ ID NOs: 1-13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 58, 60, 63, 66, 68, 70, 72, 75, 78, 81, 84, 87 or 90. 
     
     
       25. A complementary DNA strand of a nucleic acid sequence encoding the polypeptide fusion protein according to  claim 1 . 
     
     
       26. A complementary DNA strand of a nucleic acid sequence, wherein the nucleic acid sequence comprises any one of SEQ ID NOs:1-13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 58, 60, 63, 66, 68, 70, 72, 75, 78, 81, 84, 87 or 90. 
     
     
       27. A method of treating, preventing or ameliorating pain in a subject, comprising administering to said patient a therapeutically effective amount of a fusion protein according to  claim 1 . 
     
     
       28. A method according to  claim 27 , wherein the pain is chronic pain.